Replication,transcription,translation complete the central dogma of life.How mRNA,tRNA,rRNA act on ribosomes for protein synthesis.Difference between eukaryotes and prokaryotes

1. Prokaryote Gene Expression
Section 1
Overview of RNA Function

2. Overview : Section 1
 “Central Dogma” of molecular biology
 mRNA Structure and organisation
 Prokaryotic mRNA
 Eukaryotic cytoplasmic mRNA
 Eukaryotic organelle mRNA
 tRNA: structure and overview of function
 Overview of translation
 Biosynthetic cycle of mRNA
 Polycistronic and monocistronic mRNAs
 Prokaryotic and eukaryotic mRNAs

3. “Central Dogma” of molecular
biology
 “dogma” - a strongly held viewpoint or idea
 Genetic information is stored in DNA, but is
expressed as proteins, through the
intermediate step of mRNA
 The processes of Replication, Transcription
and Translation regulate this storage and
expression of information

4. Replication
 Process by which DNA (or RNA) is duplicated
from one molecule into two identical
molecules
 Semi conservative process resulting in two
identical copies each containing one parental
and one new strand of DNA
 Catalysed by DNA polymerases
 Process essentially identical between
prokaryotes and eukaryotes

5. Transcription
 Generation of single stranded RNA from a
DNA template (gene)
 Catalysed by RNA Polymerases
 Generates:
 mRNA - messenger RNA
 tRNA - transfer RNA
 rRNA - ribosomal RNA
 Occurs in prokaryotes and eukaryotes by
essentially identical processes

6. Translation
 The synthesis of a protein sequence
 Using mRNA as a template
 Using tRNAs to convert codon information
into amino acid sequence
 Catalysed by ribosomes
 Process essentially identical between
prokaryotes and eukaryotes

7. Flow of Genetic
Information
 DNA stores information in
genes
 Transcribed from
template strand into
mRNA
 Translated into protein
from mRNA by
ribosomes

8. Central Dogma
 Information in nucleic
acids (DNA or RNA)
can be replicated or
transcribed. Information
flow is reversible
 However, there is no
flow of information from
protein back to RNA or
DNA

9. Genotype and Phenotype
 A Genotype is the specific allele at a locus (gene).
Variation in alleles is the cause of variation in
individuals
 mRNA is the mechanism by which information
encoded in genes is converted to proteins
 The activities of proteins are responsible for the
phenotype attributable to a gene
 The regulation of the level of expression of mRNA is
therefore the basis for regulating the expression of
the phenotype of a gene
 Regulation is primarily at the level of varying the rate
of transcription of genes

10. mRNA Structure
 mRNAs are single stranded RNA molecules
 They are copied from the TEMPLATE strand
of the gene, to give the SENSE strand in RNA
 They are transcribed from the 5’ to the 3’ end
 They are translated from the 5’ to the 3’ end
 Generally mRNAs are linear (although some
prokaryotic RNA viruses are circular and act
as mRNAs)

11. mRNA information coding
 They can code for one or many proteins
(translation of products) in prokaryotes
(polycistronic)
 They encode only one protein (each) in
eukaryotes (monocistronic)
 Polyproteins are observed in eukaryotic
viruses, but these are a single translation
product, cleaved into separate proteins after
translation

12. RNA synthesis
 Catalysed by RNA Polymerase
 Cycle requires initiation, elongation and
termination
 Initiation is at the Promoter sequence
 Regulation of gene expression is at the
initiation stage
 Transcription factors binding to the promoter
regulate the rate of initiation of RNA
Polymerase

13. mRNA life cycle
 mRNA is synthesised by
RNA Polymerase
 Translated (once or many
times)
 Degraded by RNAses
 Steady state level depends
on the rates of both
synthesis and degradation

14. Prokaryote mRNA structure
 Linear RNA structure
 5’ and 3’ ends are unmodified
 Ribosomes bind at ribosome binding site,
internally within mRNA (do not require a free
5’ end)
 Can contain many open reading frames
(ORFs)
 Translated from 5’ end to 3’ end
 Transcribed and translated together

15. Eukaryote cytoplasmic mRNA
structure
 Linear RNA structure
 5’ and 3’ ends are modified
 5’ GpppG cap
 3’ poly A tail
 Transcribed, spliced, capped, poly
Adenylated in the nucleus, exported to
the cytoplasm

16. Eukaryote mRNA
translation
 Translated from 5’ end to 3’ end in cytoplasm
 Ribosomes bind at 5’ cap, and do require a
free 5’ end
 Can contain only one translated open reading
frames (ORF). Only first open reading frame
is translated

17. 5’ cap structures on Eukaryote
mRNA
 Caps added
enzymatically in the
nucleus
 Block degradation
from 5’ end
 Required for RNA
spicing, nuclear
export
 Binding site for
ribosomes at the
start of translation

18. Poly A tails on eukaryote
mRNA
 Added to the 3’ end by poly A polymerase
 Added in the nucleus
 Approximately 200 A residues added in a template
independent fashion
 Required for splicing and nuclear export
 Bind poly A binding protein in the cytoplasm
 Prevent degradation of mRNA
 Loss of poly A binding protein results in sudden degradation
of mRNA in cytoplasm
 Regulates biological half-life of mRNA in vivo

19. mRNA Splicing
 Eukaryote genes made up of Exons and
Introns
 mRNA transcripts contain both exons and
introns when first synthesised
 Intron sequences removed from mRNA by
Splicing in the nucleus
 Occurs in eukaryotes, but not in prokaryotes
 Alternative splicing can generate diversity of
mRNA structures from a single gene

20. Eukaryote organelle mRNA
structure
 Single stranded
 Polycistronic (many ORFs)
 Unmodified 5’ and 3’ ends
 Transcribed and translated together
 Show similarity to prokaryote genes and
transcripts

21. Transfer RNA
 Small RNAs 75 - 85 bases in length
 Highly conserved secondary and tertiary
structures
 Each class of tRNA charged with a single
amino acid
 Each tRNA has a specific trinucleotide anti-
codon for mRNA recognition
 Conservation of structure and function in
prokaryotes and eukaryotes

22. tRNA - general features
 Cloverleaf secondary
structure with constant base
pairing
 Trinucleotide anticodon
 Amino acid covalently
attached to 3’ end

23. tRNA: constant bases
and base pairing
 Constant structures of tRNAs due to
conserved bases at certain positions
 These form conserved base paired structures
which drive the formation of a stable fold
 First four double helical structures are formed
 Then the arms of the tRNA fold over to fold
the 3D structure
 The formation of triple base pairings stabilise
the overall 3D structure

24. tRNA conserved structures
 Conserved bases,
modified bases,
secondary structures
(base pairing), CAA at
3’ end
 Variable: bases,
variable loop

25. tRNA secondary structure
 Four basepaired arms
 Three single stranded
loops
 Free 3’ end
 Variable loop
 Conserved in all
Living organisms

26. tRNA 2D and 3D views
Projection of cloverleaf structure, to
ribbons outline of 3D organisation of
general tRNA structure

27. tRNA 3D ribbon - spacefill
views
Ribbon view Spacefill View

28. tRNAs have common 3D
structure
 All tRNAs have a common 3D fold
 Bind to three sites on ribosomes, which fit this
common 3D structure
 Function to bind codons on mRNA bound to
ribosome and bring amino acyl groups to the
catalytic site on the ribosome
 Ribosomes to not differentiate tRNA structure
or amino acylation.

29. Aminoacylation of tRNAs
 tRNAs have amino acids added to them by enzymes
 These enzymes are the aminoacyl tRNA synthetases
 They add the specific amino acid to the correct tRNA in an
ATP dependent charging reaction
 Each enzyme recognises a specific amino acid and its
cognate tRNA, but does not only use the anti-codon for the
specificity of this reaction
 There are 20 amino acids, 24-60 tRNAs and generally
approximately than 20 aa-tRNA synthetases

30. Information content and
tRNAs
 The information in the
mRNA in decoded by the
codon-anti-codon
interaction in ribosome
 The amino acid is not
important, as the
specificity of addition of
the amino acid is at the
charging step by the aa
tRNA synthetase

31. Ribosomes
 Highly conserved structures
 Found in all living organisms
 Made of RNA and ribosomal proteins
 Have two subunits, which bind together to
protein synthesis
 Cycle of protein synthesis consists of
Initiation, Elongation and Termination

32. Ribosome structure
 Two subunits
 50S and 30S in prokaryotes
 60S and 40S in eukaryotes
 In dynamic equilibrium
 Association in Mg2+
dependent in vitro
 In vivo cycle depends on
protein factors

33. 3D structure of ribosomes
 Most complex macromolecular complex yet
characterised
 Atomic resolution structure provides much
information about mechanisms of binding
substrates, and mechanisms of catalysis
 Is helping to clarify mechanisms of action of
antibiotics, which will lead to improved drug
designs in future

34. 50S ribosomal subunit 3D
structure

35. Overview of Translation
 Biosynthesis of polypeptide (protein)
 Requires information content from mRNA
 Catalysed by ribosomes
 Requires amino acyl-tRNAs, mRNA, various
protein factors, ATP and GTP
 Rate of translation of mRNA determined by
rate of initiation of translation of mRNA
 Translation is not generally used as a
regulatory point in control of gene expression

36. Ribosomes recycle
in protein synthesis
 Ribosomes available
in a free pool in
cytoplasm
 Bind to mRNA at
initiation of
translation
 After termination are
released from mRNA
and recycled for
further translation

37. Polysomes - one mRNA, many
ribosomes

38. Polysomes in electron
micrographs

39. Transcription and
translation
 RNA and protein synthesis are coupled processes in
prokaryotes
 As soon as the 5’ end of the mRNA is biosynthesised it is
available for translation
 Ribosomes bind, and start protein synthesis
 Degradation of the mRNA starts from the 5’ end through exo-
RNAase action
 The 5’ end can be degraded before the 3’ end is synthesised
 Coupling of these processes is important for regulation of
gene expression

40. Overall translation cycle
Elongation

41. Translation and transcription
are coupled in prokaryotes

42. Prokaryote mRNA
life cycle
 Life cycle is rapid
 Synthesis is at about 40
bases per second
 Synthesis of complete
mRNA may take 1 - 5
minutes
 Translation and degradation
occur with similar rates

43. Eukaryote mRNA lifecycle
 Transcription, capping,
polyA, splicing are nuclear
 Translation is cytoplasmic
 mRNA is complete before
export to cytoplasm (20 min
to >48 hours)
 Translation is on polysomes
 mRNA half life is 4 to > 24
hours in the cytoplasm